Project Summary: This project combines innovative technologies in eukaryotic membrane protein expression, solubilization, stablization and crystallization. An innovative protein expression system has been developed by integrating technologies that appear to overcome the major limitations for the manufacturing of mg quantities of recombinant protein, including IMPs, in mammalian cells. Stable, single cell (clonal) lines can be selected to enable controlled over-expression of protein, thereby allowing the selection of an expression system that optimizes specific activity and minimizes aggregation and/or the production of non- functional protein. A novel diagnostic technology provides the ability to rapidly determine optimal solution conditions for protein stability/solubility, reducing unwanted nonspecific aggregation. This same diagnostic can be used to map out solution conditions more likely to result in crystal formation. Specifically, we propose to: 1. Demonstrate the ability to produce milligram quantities of biologically functional membrane proteins from different membrane protein classes in mammalian cells. 2. Demonstrate the utility of a novel diagnostic technology, self-interaction chromatography (SIC), to determine second virial coefficients for different protein solvent conditions as a means to optimize co-solvent combinations and improve protein solubility and stability. 3. Demonstrate the use of SIC as an efficient, knowledge-based approach for the production of diffraction-quality crystals of membrane proteins. The combination of these technologies is expected to improve success rates for determining structures of integral membrane proteins. This project directly addresses several of the current impediments to membrane protein structural biology (outlined on pages 3-6 of this RFA) including protein production, stability, solubility and crystallization. The project involves collaborations with more than fifteen NIH-funded investigators studying different biologically and medically relevant membrane proteins. The project will not only support structural studies performed by the PI, but also provide protein and crystals to other individual crystallographers, NMR spectroscopists and one membrane protein "Specialized Center". Relevance: This work will directly support future efforts to develop new therapeutics as well as enhancing our knowledge of membrane biology. Membrane proteins account for 70% of the drugable market, addressing a wide range of therapeutic categories including pain, asthma, inflammation, obesity, cancer, cardiovascular, metabolic, gastrointestinal and central nervous system diseases cystic fibrosis and more.